Bacterium for the production of an exopolysaccharide comprising N-acetylglucosamine and D-glucose

ABSTRACT

The present disclosure is directed to microorganisms that are genetically modified to express at least one exogenous enzyme involved in N-acetylglucosamine uptake and metabolism. Methods for the production of an exopolysaccharide are also disclosed. The exopolysaccharide comprises N-acetylglucosamine and D-glucose and may be used as a bioresorbable implant for soft tissue repair, replacement, or augmentation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/467,917 filed Mar. 7, 2017, the disclosure of which is herebyincorporated by reference in its entirety.

TECHNICAL FIELD

This disclosure relates to genetically-modified microorganisms andmethods for the production of exopolysaccharides comprisingN-acetylglucosamine and D-glucose monomers.

BACKGROUND

Cellulose produced in bacteria has several advantages compared toplant-derived cellulose for human health applications. Bacterialcellulose has high strength, excellent conformability and handling, goodbiocompatibility, and is free of lignins and other undesirable materialsoften found in plant cellulose. Because of these properties, bacterialcellulose is an attractive biomaterial for use in human health. In somehuman health applications, it is preferred that the material isbioresorbed as, for example, native human tissue is regenerated at thesite where the bacterial cellulose has been placed. Because humans lackthe enzymes capable of degrading cellulose, however, bacterial celluloseis not biodegradable in humans. Although various postproduction chemicalprocesses have been developed to modify cellulose to make it susceptibleto hydrolysis and hence, bioresorbable, these chemical processes canresult in changes to the properties of the resulting material and lossof some of the desirable features of bacterial cellulose.

SUMMARY

Disclosed herein are microorganisms for the production of anexopolysaccharide wherein the microorganism expresses an exogenousenzyme. In one embodiment, the exogenous enzyme is N-acetylglucosaminephosphoenolpyruvate-dependent sugar phosphotransferase system permeaseor a biologically active homologue thereof. The microorganism producesan exopolysaccharide that comprises N-acetylglucosamine and D-glucose.

According to one embodiment, the microorganism further expresses atleast one exogenous enzyme involved in N-acetylglucosamine uptake ormetabolism. For example, the at least one exogenous enzyme involved inN-acetylglucosamine uptake or metabolism may comprise one or more ofN-acetylglucosamine kinase, phosphoacetyl-glucosamine mutase, andUDP-N-acetylglucosamine pyrophosphorylase. Accordingly, in someembodiments, the microorganism further expresses at least one exogenousenzyme comprising one or more of N-acetylglucosamine kinase,phosphoacetyl-glucosamine mutase, and UDP-N-acetylglucosaminepyrophosphorylase. In preferred embodiments, the N-acetylglucosaminekinase and/or the UDP-N-acetylglucosamine pyrophosphorylase are derivedfrom a source other than yeast.

Also provided herein are bacteria for the production of anexopolysaccharide wherein the bacteria expresses at least one exogenousenzyme, wherein the at least one exogenous enzyme is N-acetylglucosaminekinase, yeast-derived phosphoacetyl-glucosamine mutase, orUDP-N-acetylglucosamine pyrophosphorylase. The bacterium produces anexopolysaccharide that comprises N-acetylglucosamine and D-glucose. TheN-acetylglucosamine kinase and the UDP-N-acetylglucosaminepyrophosphorylase may be derived from a source other than yeast, or theN-acetylglucosamine kinase may be derived from a source other than yeastand the UDP-N-acetylglucosamine pyrophosphorylase may be derived fromyeast, or the N-acetylglucosamine kinase may be derived from yeast andthe UDP-N-acetylglucosamine pyrophosphorylase may be derived from asource other than yeast.

In certain embodiments, the exogenous enzyme is encoded by a nucleicacid. In some embodiments, the nucleic acid can be a cDNA molecule.According to one embodiment, the nucleic acid is contained in arecombinant vector. For example, the recombinant vector may be a viralor a plasmid vector. According to a further embodiment, the nucleic acidis operably linked to a promoter. For example, the promoter may be aconstitutive or inducible promoter.

Further disclosed are methods for producing exopolysaccharides that,according to one embodiment, comprises culturing a microorganism underconditions effective to produce the exopolysaccharide and recovering theexopolysaccharide. The microorganism expresses an exogenous enzyme,wherein the exogenous enzyme is N-acetylglucosaminephosphoenolpyruvate-dependent sugar phosphotransferase system permeaseor a biologically active homologue thereof. The microorganism producesan exopolysaccharide that comprises N-acetylglucosamine and D-glucose.

According to one embodiment, the microorganism further expresses atleast one exogenous enzyme involved in N-acetylglucosamine uptake and/ormetabolism. For example, the at least one exogenous enzyme involved inN-acetylglucosamine uptake or metabolism may comprise one or more ofN-acetylglucosamine kinase, phosphoacetyl-glucosamine mutase, andUDP-N-acetylglucosamine pyrophosphorylase. According, in someembodiments, the microorganism further expresses at least one exogenousenzyme comprising one or more of N-acetylglucosamine kinase,phosphoacetyl-glucosamine mutase, or UDP-N-acetylglucosaminepyrophosphorylase. In some aspects, the N-acetylglucosamine kinaseand/or the UDP-N-acetylglucosamine pyrophosphorylase are derived from asource other than yeast.

Methods for producing an exopolysaccharide are also provided, whereinthe methods comprise culturing a microorganism under conditionseffective to produce the exopolysaccharide and recovering theexopolysaccharide. The microorganism expresses at least one exogenousenzyme, wherein the at least one exogenous enzyme is N-acetylglucosaminekinase, yeast-derived phosphoacetyl-glucosamine mutase, orUDP-N-acetylglucosamine pyrophosphorylase, wherein the exopolysaccharidecomprises N-acetylglucosamine and D-glucose. The N-acetylglucosaminekinase and the UDP-N-acetylglucosamine pyrophosphorylase may be derivedfrom a source other than yeast, or the N-acetylglucosamine kinase may bederived from a source other than yeast and the UDP-N-acetylglucosaminepyrophosphorylase may be derived from yeast, or the N-acetylglucosaminekinase may be derived from yeast and the UDP-N-acetylglucosaminepyrophosphorylase may be derived from a source other than yeast.

The microorganism described herein may be a bacterium. In someembodiments, the bacterium belongs to the family Acetobacteraceae.Exemplary bacterium from the family Acetobacteraceae include those ofthe genus Komagataeibacter and Gluconacetobacter. In some embodiments,the bacterium belongs to a genus comprising Agrobacterium, Aerobacter,Achromobacter, Azotobacter, Rhizobium, Sarcina, or Salmonella. Inpreferred embodiments, the bacterium is Komagataeibacter xylinus.

The microorganism may be cultured in a culture medium containingD-glucose, N-acetylglucosamine, glucosamine, 3-O-methylglucose,2-deoxyglucose, or a combination thereof.

Also described herein are methods of treating the exopolysaccharideafter it is produced by the microorganism. In some embodiments, theexopolysaccharide may be at least partially dehydrated by, for example,mechanical pressing and/or critical point drying using supercriticalcarbon dioxide. In some embodiments, the exopolysaccharide may becontacted with one or more active agents. In some embodiments, theexopolysaccharide may be further reacted with an oxidizing agent so asto form a body of oxidized exopolysaccharide. For example, the oxidizingagent may comprise metaperiodate, hypochlorite, dichromate, peroxide,permanganate, or nitrogen dioxide. In some embodiments, the oxidizingagent is sodium metaperiodate.

The present disclosure further describes implants for soft tissuerepair, replacement, or augmentation comprising the exopolysaccharideproduced by any of the methods described herein. In preferredembodiments, the implant is used in hernia repair, repair of dura mater,replacement of dura mater, or any combination thereof.

DETAILED DESCRIPTION

It is to be understood that the disclosed microorganisms and methods arenot limited to the specific microorganisms and methods described and/orshown herein, and that the terminology used herein is for the purpose ofdescribing particular embodiments by way of example only and is notintended to be limiting of the claimed microorganisms and methods.

Unless specifically stated otherwise, any description as to a possiblemechanism or mode of action or reason for improvement is meant to beillustrative only, and the disclosed microorganisms and methods are notto be constrained by the correctness or incorrectness of any suchsuggested mechanism or mode of action or reason for improvement.

Where a range of numerical values is recited or established herein, therange includes the endpoints thereof and all the individual integers andfractions within the range, and also includes each of the narrowerranges therein formed by all the various possible combinations of thoseendpoints and internal integers and fractions to form subgroups of thelarger group of values within the stated range to the same extent as ifeach of those narrower ranges was explicitly recited. Where a range ofnumerical values is stated herein as being greater than a stated value,the range is nevertheless finite and is bounded on its upper end by avalue that is operable within the context of the invention as describedherein. Where a range of numerical values is stated herein as being lessthan a stated value, the range is nevertheless bounded on its lower endby a non-zero value. It is not intended that the scope of the inventionbe limited to the specific values recited when defining a range. Allranges are inclusive and combinable.

When values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms anotherembodiment. Reference to a particular numerical value includes at leastthat particular value, unless the context clearly dictates otherwise.

It is to be appreciated that certain features of the disclosedmicroorganisms and methods which are, for clarity, described herein inthe context of separate embodiments, may also be provided in combinationin a single embodiment. Conversely, various features of the disclosedmicroorganisms and methods that are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany subcombination.

As used herein, the singular forms “a,” “an,” and “the” include theplural.

Various terms relating to aspects of the description are used throughoutthe specification and claims. Such terms are to be given their ordinarymeaning in the art unless otherwise indicated. Other specificallydefined terms are to be construed in a manner consistent with thedefinitions provided herein.

The term “about” when used in reference to numerical ranges, cutoffs, orspecific values is used to indicate that the recited values may vary byup to as much as 10% from the listed value. Thus, the term “about” isused to encompass variations of ±10% or less, variations of ±5% or less,variations of ±1% or less, variations of ±0.5% or less, or variations of±0.1% or less from the specified value.

The term “comprising” is intended to include examples encompassed by theterms “consisting essentially of” and “consisting of”; similarly, theterm “consisting essentially of” is intended to include examplesencompassed by the term “consisting of.”

The term “exogenous” refers to any protein that does not originate fromthat particular cell as found in nature. Accordingly,non-naturally-occurring proteins are considered to be exogenous to acell once introduced into the cell.

Because bacterial cellulose is not biodegradable in humans, there existsa need to produce a bioresorbable exopolysaccharide that exhibits thedesired features of bacterial cellulose. One approach to generate such abioresorbable exopolysaccharide is to modify a bacterium that producescellulose such that chemical linkages that are susceptible to cleavageby human enzymes are incorporated into the exopolysaccharide duringproduction. Bacterial cellulose is a polysaccharide comprised ofβ(1-4)-linked D-glucose monomers. These units can be cleaved bycellulase enzymes; however, these enzymes are not present in humans andconsequently bacterial cellulose is not biodegradable in humans.N-acetylglucosamine (GlcNac) is a naturally occurring monosaccharide.Polysaccharides comprised of β(1-4)-linked GlcNac residues, such aschitin, are substrates for human lysozyme enzymes and consequently arebiodegradable in humans. Incorporation of β(1-4)-linked GlcNac residuesinstead of β(1-4)-linked D-glucose into an exopolysaccharide produced inbacteria results in an exopolysaccharide where some number of theglucose units are replaced by GlcNac units, and is susceptible to humanlysozyme and therefore bioresorbable in humans.

Efficient incorporation of GlcNac residues into a bacterialexopolysaccharide can depend on the appropriate amount of GlcNacmonomers in an appropriate chemical form present intracellularly in theproducing bacteria so that the GlcNac monomers will be utilized as asubstrate by the bacterial cellulose biosynthetic machinery. Utilizationby the cellulose biosynthesis machinery of the GlcNac substrate incompetition with the natural glucose substrate results in aheterogeneous exopolysaccharide where biodegradable chemical linkagesare substituted for a portion of the β(1-4) glucose linkages. Forcellulose production in Komagataeibacter xylinus (K. xylinus), forexample, the uridine diphosphate monosaccharide form of the monomer(either UDP-glucose or UDP-GlcNac) is utilized as substrate for thecellulose synthase enzyme. In turn, UDP-GlcNac is produced from thereaction of GlcNac-1-phosphate and UTP catalyzed by aUDP-GlcNac-pyrophosphorylase. GlcNac-1-phosphate is made byisomerization of GlcNac-6-phosphate catalyzed byphosphoacetyl-glucosamine mutase.

The sequence outlined above starts with the efficient accumulation ofGlcNac-6-phosphate intracellularly in the bacterium. Bacterialphosphoenolpyruvate:GlcNac phosphotransferase systems mediate thetransport of GlcNac intracellularly from the extracellular milieu, whichalso results in the simultaneous phosphorylation of GlcNac at the 6position to form GlcNac-6-phosphate. Thus, genetically modifying K.xylinus to overexpress a GlcNac phosphotransferase results in both theefficient transport of GlcNac into the cell and its conversion to the 6phosphate form that is the starting point for ultimate incorporationinto a cellulose-based polysaccharide. Genetically engineeredmicroorganisms, including bacteria, and methods of producingexopolysaccharides are provided herein.

Microorganisms for the Production of Exopolysaccharides

Provided herein are microorganisms for the production ofexopolysaccharides wherein the microorganisms express an exogenousenzyme. Suitable microorganisms include, but are not limited to,bacteria and fungi. In one embodiment, the fungi can be a yeastincluding, but not limited to, a yeast of the genus Saccharomyces,Candida, Hansenula, Pichia, Kluveromyces, Phaffia, andSchizosaccharomyces. Suitable yeast include, but are not limited to,yeast from the species selected from Saccharomyces cerevisiae,Schizosaccharomyces pombe, Candida albicans, Hansenula polymorpha,Pichia pastoris, P. canadensis, Kluyveromyces marxianus and Phaffiarhodozyma. In another embodiment, the microorganism can be a fungusincluding, but not limited to, a fungus of the genus Aspergillus,Absidia, Rhizopus, Chrysosporium, Neurospora and Trichoderma. Suitablefungi include, but are not limited to, fungi from the species selectedfrom Aspergillus niger, A. nidulans, Absidia coerulea, Rhizopus oryzae,Chrysosporium lucknowense, Neurospora crassa, Nintermedia and Trichodermreesi. In a further embodiment, the microorganism can be a bacterium. Insome embodiments, the bacterium belongs to the family Acetobacteraceae,including those of the genus Komagataeibacter and Gluconacetobacter. Insome embodiments, the bacterium belongs to the genus Escherichia,Bacillus, Lactobacillus, Pseudomonas, Streptomyces, Agrobacterium,Aerobacter, Achromobacter, Azotobacter, Rhizobium, Sarcina, orSalmonella. Preferably, the bacteria belong to the genusKomagataeibacter, Gluconacetobacter, Agrobacterium, Aerobacter,Achromobacter, Azotobacter, Rhizobium, Sarcina, or Salmonella. Suitablebacteria include, but are not limited to, bacteria from the speciesselected from Escherichia coli, Bacillus subtilis, Bacilluslicheniformis, Lactobacillus brevis, Pseudomonas aeruginosa,Streptomyces lividans, Gluconacetobacter xylinus and Komagataeibacterxylinus. Most preferably, the bacterium belongs to the speciesKomagataeibacter xylinus.

The microorganism produces an exopolysaccharide that comprisesN-acetylglucosamine and D-glucose. As used herein, “exopolysaccharide”refers to a polymer of glucose and glucose analog residues secreted by amicroorganism such as a bacterium. In some aspects, theexopolysaccharide may be microbial cellulose ((1-4)-linked-β-D-glucan),which is composed of monomers bound by β(1-4) linkages with the chemicalformula (C₆H₁₀O₅)_(n). Glucose, also referred to as D-Glucose,(2R,3S,4R,5R)-2,3,4,5,6-Pentahydroxyhexanal or Glc, has the chemicalformula C₆H₁₂O₆ and the Chemical Abstracts Service registry number50-99-7. According to another aspect, the exopolysaccharide may bechitin ((1-4)-linked 2 acetoamino-2-deoxy-β-D-glucose), which iscomposed of N-acetylglucosamine monomers bound by β(1-4) linkages withthe chemical formula (C₈H₁₃O₅N)_(n). Chitin may also exist as acopolymer of N-acetylglucosamine and N-glucosamine residues.N-acetylglucosamine, also referred to asβ-D-(Acetylamino)-2-deoxy-glucopyranose, N-acetyl-D-glucosamine, GlcNAC,or NAG, has the chemical formula (C₆H₁₅NO₆) and the Chemical AbstractsService registry number 7512-17-6. In preferred embodiments, theexopolysaccharide may be a copolymer, meaning a polymer composed of atleast two different monomer subunits. In most preferred embodiments, theexopolysaccharide comprises cellulose-chitin(glucose:N-acetylglucosamine) copolymers. A mixture of different molarratios of glucose to N-acetylglucosamine may be synthesized by thedisclosed methods.

In one embodiment, the microorganism expresses the exogenous enzymeN-acetylglucosamine phosphoenolpyruvate-dependent sugarphosphotransferase system permease or a biologically active homologuethereof. N-acetylglucosamine phosphoenolpyruvate-dependent sugarphosphotransferase system permease (EII^(Nag)) is a type II enzymeencoded by the gene nagE, which has been sequenced in E. coli (EcoGeneEG10635) and K. pneumoniaei (GenBank X63289). EII^(Nag) is a componentof the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system(PTS), which is involved in the transport and phosphorylation of a largenumber of carbohydrates. In particular, EII^(Nag) is a multidomain,membrane-spanning protein responsible for the phosphorylation ofN-acetylglucosamine at the 6 position to formN-acetylglucosamine-6-phosphate while the N-acetylglucosamine is beingtransported across the inner membrane. EII^(Nag) may also be referred toas N-acetylglucosamine PTS permease, II^(Nag), pLC5-21, GlcNAc-specificenzyme II, or IIN-acetylglucosamine. Expression or overexpression of theEII^(Nag) protein will result in the production of an exopolysaccharidethat comprises N-acetylglucosamine and D-glucose monomers.

As used herein, the term “express” refers to the process by whichinformation from a gene is first converted into messenger RNA(transcription) and then to a functional gene product such as proteins(translation) as well as non-protein coding genes such as transfer RNAor small nuclear RNA. “Overexpression” refers to an increased frequencyof expression relative to endogenous levels.

The microorganism can further express at least one exogenous enzymeinvolved in N-acetylglucosamine uptake or metabolism. The at least oneexogenous enzyme involved in N-acetylglucosamine uptake or metabolismmay comprise one or more of N-acetylglucosamine kinase,phosphoacetyl-glucosamine mutase, or UDP-N-acetylglucosaminepyrophosphorylase. Accordingly, in some embodiments, the microorganismcan further express at least one exogenous enzyme comprising one or moreof N-acetylglucosamine kinase, phosphoacetyl-glucosamine mutase, orUDP-N-acetylglucosamine pyrophosphorylase. In other preferredembodiments, the N-acetylglucosamine kinase and/or theUDP-N-acetylglucosamine pyrophosphorylase are derived from a sourceother than yeast.

In some embodiments, the exogenous enzyme can be encoded by a nucleicacid. Suitable nucleic acids include, for example, cDNA molecules. Insome aspects, the nucleic acid is contained in a recombinant vector. Therecombinant vector can be a viral or a plasmid vector. The nucleic acidcan be operably linked to a promoter, such as a constitutive orinducible promoter.

Provided herein are bacteria for the production of an exopolysaccharidewherein the bacteria expresses at least one exogenous enzyme, whereinthe at least one exogenous enzyme is N-acetylglucosamine kinase,yeast-derived phosphoacetyl-glucosamine mutase, orUDP-N-acetylglucosamine pyrophosphorylase. The bacteria produce anexopolysaccharide that comprises N-acetylglucosamine and D-glucose. TheN-acetylglucosamine kinase and the UDP-N-acetylglucosaminepyrophosphorylase may be derived from a source other than yeast, or theN-acetylglucosamine kinase may be derived from a source other than yeastand the UDP-N-acetylglucosamine pyrophosphorylase may be derived fromyeast, or the N-acetylglucosamine kinase may be derived from yeast andthe UDP-N-acetylglucosamine pyrophosphorylase may be derived from asource other than yeast.

N-acetylglucosamine kinase (Chemical Abstracts Service Number 9027-48-9)is an enzyme that catalyzes the conversion of N-acetylglucosamine toN-acetylglucosamine-6-phosphate. N-acetylglucosamine kinase, alsoreferred to as GlcNAc kinase, N-acetylglucosamine kinase, NAG5, NAGK,and N-acetyl-D-glucosamine-6-phosphotransferase, belongs to a family oftransferases implicated in the transfer of phosphorous-containing groupsto an alcohol acceptor. Accordingly, N-acetylglucosamine kinase works inparallel with EII^(Nag) to generate N-acetylglucosamine-6-phosphate,which may be used as a substrate for further downstream enzymaticreactions.

Phosphoacetyl-glucosamine mutase (Chemical Abstracts Service Number9027-51-4) is an enzyme that belongs to the phosphohexose mutase family,which catalyzes the conversion of N-acetylglucosamine-6-phosphate toN-acetylglucosamine-1-phosphate. Phosphoacetyl-glucosamine mutase isalso known as N-acetyl-alpha-D-glucosamine 1,6-phosphomutase,acetylglucosamine phosphomutase, acetylglucosamine phosphomutase,acetylaminodeoxyglucose phosphomutase, phospho-N-acetylglucosaminemutase, AGM1, PCM1, PGM3, and N-acetyl-D-glucosamine 1,6-phosphomutase.In preferred embodiments, phosphoacetyl-glucosamine mutase is derivedfrom yeast such as, for example, the species C. albicans or S.cerevisiae. The C. albicans phosphoacetyl-glucosamine mutase is encodedby the agm1 gene. The S. cerevisiae the phosphoacetyl-glucosamine mutaseis encoded by the pcm1 gene.

UDP-N-acetylglucosamine pyrophosphorylase (Chemical Abstracts ServiceNumber 9023-06-7) catalyzes the conversion ofN-acetylglucosamine-1-phosphate to UDP-N-acetylglucosamine.UDP-N-acetylglucosamine pyrophosphorylase is also referred to asUTP:N-acetyl-alpha-D-glucosamine-1-phosphate uridylyltransferase,UDP-GlcNAc pyrophosphorylase, UAP1, and acetylglucosamine 1-phosphateuridylyltransferase. UDP-N-acetylglucosamine pyrophosphorylase is amember of the family of nucleotide diphosphate sugar pyrophosphorylases,comprising a subfamily of UDP-sugar pyrophosphorylases. In Escherichiacoli, the glmU gene encodes for a bifunctional UDP-N-acetylglucosaminepyrophosphorylase/Glucosamine-1-phosphate N-acetyltransferase(EC:2.7.7.23 and 2.3.1.157) enzyme that catalyzes the last twosequential reactions in the de novo biosynthetic pathway forUDP-N-acetylglucosamine (UDP-GlcNAc): 1) the C-terminal domain catalyzesthe transfer of an acetyl group from acetyl coenzyme A toglucosamine-1-phosphate (GlcN-1-P) to produceN-acetylglucosamine-1-phosphate (GlcNAc-1-P); and 2) the N-terminaldomain catalyzes the transfer of a uridine 5-monophosphate (from uridine5-triphosphate) to the N-acetylglucosamine-1-phosphate to formUDP-GlcNAc. In some embodiments, the UDP-N-acetylglucosaminepyrophosphorylase is encoded by the Escherichia coli glmU gene.

In certain embodiments, the exogenous enzyme is encoded by a nucleicacid. The term “nucleic acid” refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analogs ofnatural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless specifically limited otherwise,the term also refers to oligonucleotide analogs including PNA(peptidonucleic acid), analogs of DNA used in antisense technology(e.g., phosphorothioates, phosphoroamidates). Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (including but notlimited to, degenerate codon substitutions) and complementary sequencesas well as the sequence explicitly indicated. Specifically, degeneratecodon substitutions are achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues.

In some embodiments, the nucleic acid is a cDNA molecule.

The nucleic acids can be contained in a recombinant vector such as, forexample, plasmids, phage, phagemids, viruses, artificial chromosomes andthe like. In some embodiments, the recombinant vector is a viral orplasmid vector. Preferably, the vector is capable of replicatingautonomously within the microorganism. Alternatively, the vector mayintegrate into the host's genome. More preferably, the vector is anexpression vector. The nucleic acid may be operably linked to apromoter. More than one gene may be operably linked to a singlepromoter. The term “operably linked” means a functional linkage betweena nucleic acid expression control sequence such as a promoter and asecond nucleic acid sequence, wherein the expression control sequenceaffects transcription and/or translation of the nucleic acidcorresponding to the second sequence. In some embodiments, the promoteris a constitutive or inducible promoter.

The coding region of the gene may be altered prior to insertion into orwithin the recombinant vector such as by deletion, addition orsubstitution. In certain embodiments, it may be desirable to alter thefunction or specificity of the enzyme. Additionally, the coding regionsof two or more enzymes may be linked to create a fusion protein.

The bacterium can belong to the family Acetobacteraceae or a genuscomprising Komagataeibacter, Gluconacetobacter, Agrobacterium,Aerobacter, Achromobacter, Azotobacter, Rhizobium, Sarcina, andSalmonella. In some embodiments, the bacterium is Komagataeibacterxylinus.

Methods of Producing Exopolysaccharides

The present disclosure additionally describes methods for producingexopolysaccharides comprising culturing a microorganism under conditionseffective to produce the exopolysaccharide and recovering theexopolysaccharide. The microorganism expresses an exogenous enzyme,wherein the exogenous enzyme can be N-acetylglucosaminephosphoenolpyruvate-dependent sugar phosphotransferase system permeaseor a biologically active homologue thereof. The exopolysaccharidecomprises N-acetylglucosamine and D-glucose.

The microorganism can further express at least one exogenous enzymeinvolved in N-acetylglucosamine uptake and metabolism. The at least oneexogenous enzyme involved in N-acetylglucosamine uptake and metabolismcan comprise one or more of N-acetylglucosamine kinase,phosphoacetyl-glucosamine mutase, or UDP-N-acetylglucosaminepyrophosphorylase. In some aspects, the N-acetylglucosamine kinaseand/or the UDP-N-acetylglucosamine pyrophosphorylase are derived from asource other than yeast.

In some aspects of the disclosed methods, the microorganism is abacterium. Thus, the methods for producing an exopolysaccharide cancomprise culturing a bacterium under conditions effective to produce theexopolysaccharide, and recovering the exopolysaccharide.

Exemplary bacterium are those belonging to a genus comprisingKomagataeibacter, Gluconacetobacter, Agrobacterium, Aerobacter,Achromobacter, Azotobacter, Rhizobium, Sarcina, and Salmonella. In someembodiments, for example, the bacterium can be Komagataeibacter xylinus.

In embodiments wherein the microorganism is a bacterium, the at leastone exogenous enzyme can be N-acetylglucosamine kinase, yeast-derivedphosphoacetyl-glucosamine mutase, or UDP-N-acetylglucosaminepyrophosphorylase. The N-acetylglucosamine kinase and theUDP-N-acetylglucosamine pyrophosphorylase are derived from a sourceother than yeast, or the N-acetylglucosamine kinase is derived from asource other than yeast and the UDP-N-acetylglucosaminepyrophosphorylase is derived from yeast, or the N-acetylglucosaminekinase is derived yeast and the UDP-N-acetylglucosaminepyrophosphorylase is derived from a source other than yeast.

In the above disclosed methods for producing an exopolysaccharide, themicroorganism (including the bacterium) can be cultured in a culturemedium containing D-glucose, N-acetylglucosamine, glucosamine,3-O-methylglucose, 2-deoxyglucose, or a combination thereof.

In preparing the exopolysaccharides, microorganisms such asKomagataeibacter xylinus (Acetobacter xylinum) expressing one or moreexogenous enzymes as presently described are cultured (incubated) in abioreactor containing a liquid nutrient medium at about 30° C. at aninitial pH of about 4.1 to about 4.5. Exopolysaccharide production canbe achieved using, for example, sucrose, glucose, N-acetylglucosamine,glucosamine, 3-O-methylglucose, and/or 2-deoxyglucose as a carbonsource, ammonium salts as a nitrogen source, and corn steep liquor asnutrient source.

Suitable bioreactors are selected which minimize evaporation and provideadequate oxygen-limiting conditions. The bioreactor may be composed of aclean, dry plastic box fitted with an airtight or limited gas-permeablecover. An aeration port can be added to regulate the oxygen-limitingconditions. Dimensions of the bioreactor can vary in configurationdepending on the desired shape, size, thickness, and yield of theexopolysaccharide being produced. Such systems are able to provideoxygen-limiting conditions that help ensure formation of a uniformexopolysaccharide membrane.

The main fermentation process, following the incubation step, istypically carried out under stationary conditions for a period of about8-120 hours, preferably 24-72 hours, during which the bacteria in theculture medium synthesize and deposit thin layers of exopolysaccharidesheets, thus forming an exopolysaccharide membrane (pellicle). Dependingon the desired thickness and/or exopolysaccharide yield, thefermentation can be stopped, at which point the membrane can beharvested from the bioreactor. According to one embodiment, the mainfermentation is stopped after a relatively short period to yield auniform, low exopolysaccharide content membrane. The excess mediumcontained in the pellicle is then removed by standard separationtechniques such as compression or centrifugation, which results in apartially dehydrated pellicle.

The partially dehydrated exopolysaccharide pellicle can then besubjected to a purification processing that renders theexopolysaccharide nonpyrogenic. According to one embodiment, thepurification method is a chemical purification of the exopolysaccharidemembrane. The exopolysaccharide can be subjected to a series of caustic(e.g., concentrated sodium hydroxide) chemical wash steps to convert theexopolysaccharide membrane into a nonpyrogenic material, followed bysoaking and/or rinsing with filtered water, until a neutral pH isachieved. Alternatively, or in conjunction with these steps, a shortsoak in diluted acetic acid can also be conducted to ensureneutralization of the remaining sodium hydroxide. Purification processesusing various exposure times, concentrations and temperatures, as wellas mechanical techniques including pressing, can be utilized on eitherthe purified or unpurified exopolysaccharide membrane. Processing timesin sodium hydroxide of about 1 hour to about 12 hours have been studiedin conjunction with temperature variations of about 30° C. to about 100°C. to optimize the process. A preferred or recommended temperatureprocessing occurs at or near 70° C.

The amount of endotoxins left in the exopolysaccharide after processingmay be measured by Limulus Amebocyte Lysate (LAL) test. The cleaningprocess described herein is capable of providing a nonpyrogenicexopolysaccharide membrane (<0.06 EU/ml). Following the purification ofthe exopolysaccharide membrane, according to one embodiment, thepellicle can be mechanically compressed to a desired weight andthickness.

The exopolysaccharide membrane can undergo critical point drying.Critical point drying is a stepwise process wherein water in theexopolysaccharide membrane is exchanged with a non-aqueous solvent thatis soluble with water, for example ethanol. The ethanol is thendisplaced with liquid carbon dioxide. This drying process can enhancethe penetration of the oxidizing agent into the exopolysaccharidemembrane. The dried membrane can be further reacted with an oxidizingagent, as described below, and recovered and washed in a manner asdescribed above.

In another embodiment, the exopolysaccharide may be at least partiallydehydrated by, for example, mechanical pressing. Following thepurification and/or oxidations steps described above, theexopolysaccharide pellicle is mechanically compressed to thepredetermined weight desired for form, fit, and function for soft tissuerepair, replacement, or augmentation, e.g., hernia repair. The originalfill volume and the compression steps are integral to attain the desireddensity that affects the strength, integrity, and function of theexopolysaccharide. Partially dehydrated samples are packaged in asingle- or double-pouch system in preparation for sterilization. Samplesare tested for exopolysaccharide content, endotoxin, and mechanicalstrength.

Further processing may continue with placing the exopolysaccharide in aclosed container and decreasing the temperature to below 0° C. After aperiod of time, the temperature is increased to above freezing andexcess moisture that is released from the exopolysaccharide is removed.This results in partially dehydrated exopolysaccharide. Without beingbound to any one theory, it is believed that at below 0° C., watercrystals form and are brought to the surface of the exopolysaccharidemesh. At above freezing temperature the liquid that has been removed isnot allowed to rehydrate the surgical mesh, thereby yielding a producthaving increased tensile strength, elongation (stretch), conformability,and suture retention when used as an implantable medical device forvarious surgical procedures. Depending on the desired level ofdehydration, the films are exposed to one or more temperature variationcycles. The excess liquid is removed by pouring, dabbing, or vacuumingit off. The partially dehydrated material is packaged in a single- ordouble-pouch system in preparation for sterilization. Samples are testedfor exopolysaccharide content, endotoxin (LAL), and mechanical strength.

The exopolysaccharide may be at least partially dehydrated by criticalpoint drying using supercritical carbon dioxide. Thus, in someembodiments, the disclosed methods can further comprise the step of atleast partially dehydrating the body of the exopolysaccharide bycritical point drying using supercritical carbon dioxide. As previouslyexplained above, the water in the exopolysaccharide membrane isexchanged with a non-aqueous solvent (e.g., ethanol). The solvent isthen replaced with liquid carbon dioxide through a process calledcritical point drying. During critical point drying, theexopolysaccharide membranes are loaded onto a holder, sandwiched betweenstainless steel mesh plates, and then soaked in a chamber containingsupercritical carbon dioxide under pressure. The holder is designed toallow the CO₂ to circulate through the exopolysaccharide membrane whilemesh plates stabilize the membrane to prevent the membrane from wavingduring the drying process. Once all of the organic solvent has beenremoved (which in most typical cases is in the range of about 1-6hours), the liquid CO₂ temperature is increased above the criticaltemperature for carbon dioxide so that the CO₂ forms a supercriticalfluid/gas. Due to the fact that no surface tension exists during suchtransition, the resulting product is a dried membrane which maintainsits shape, thickness, and 3-D nanostructure. The dried product undergoescutting, packaging, and sterilization.

The exopolysaccharides produced from the disclosed methods can befurther contacted with one or more active agents. Suitable active agentsinclude any compositions suitable for treatment at an anatomicallocation, such as, bone marrow, autograft, osteoinductive smallmolecules, osteogenic material, stem cells, bone morphogenetic proteins,antibacterial agents, calcium phosphate ceramics, and mixtures andblends thereof.

The exopolysaccharide can be reacted with an oxidizing agent. Suitableoxidizing agents include, for example, metaperiodate, hypochlorite,dichromate, peroxide, permanganate, or nitrogen dioxide. In someembodiments, the oxidizing agent is sodium metaperiodate. The oxidizingagent can have, according to one embodiment, a concentration range ofabout 0.01M to about 10.0M, preferably about 0.05M to about 1.0M, andmore preferably from about 0.1M to about 0.5M. It should be noted thatwhen selecting metaperiodate, the reaction is preferably conducted inthe dark. According to one embodiment, the oxidation reaction with theoxidizing agent is for a time period in the range of about 30 minutes to72 hours, preferably about 2-16 hours, and more preferably about 2-6hours. The oxidation reaction can typically proceed at a temperaturerange of 18° C. to 60° C., preferably 30° C. to 50° C., and morepreferably at about 40° C. According to another embodiment, theoxidation reaction with the oxidizing agent is for a time period of atleast about one hour, and in yet another embodiment for at least about 3hours. The container(s) are placed on a shaker and agitated at 20-500rpm, preferably 350-450 rpm. The molar ratio between exopolysaccharideand metaperiodate can be maintained at the range of 1:1-1:160,preferably 1:1-1:120, and more preferably at about 1:120. Uponcompletion of the oxidation reaction, the oxidized exopolysaccharidemembrane can be washed multiple times in filtered water on an ice-bathto remove excess metaperiodate. Alternatively, it can be washed inethylene glycol to neutralize metaperiodate followed by multiple rinsesin DI water.

The oxidized exopolysaccharide can have a variable range of degree ofoxidation, which can, according to one embodiment, be in the range ofabout 0 percent to about 99 percent oxidation, for example in a range ofabout 20 percent to about 70 percent. The degree of oxidation of theirradiated oxidized exopolysaccharide can depend on the oxidizing agentselected, the concentration range of the oxidizing agent, reactiontemperature, and the time period of the reaction between the irradiatedexopolysaccharide and the oxidizing agent. According to one embodiment,the degree of oxidation is in the range of about 15 percent to about 80percent, and in another embodiment is in the range of about 20 percentto about 70 percent. In addition, the exopolysaccharide membrane can beground up to form a slurry and then homogenized into a fine suspensionof fibers. The suspension can then be placed in a mold and cross-linkedto form a stable exopolysaccharide membrane again.

Oxidation may be performed after the exopolysaccharide undergoescritical point drying, partial dehydration, and/or contact with one ormore active agents.

Exopolysaccharide Implants

Also provided are exopolysaccharide products produced by the processesdisclosed herein. In particular, the present disclosure furtherdescribes implants for soft tissue repair, replacement, or augmentationcomprising the exopolysaccharide produced by any of the methodsdescribed herein. The implant can be used in hernia repair, repair ofdura mater, replacement of dura mater, or any combination thereof.According to the present disclosure, an implant is described havingsufficient mechanical strength, conformability to anatomical surfaces,and resorption profile for use in tissue repair, replacement, and/oraugmentation procedures, particularly soft tissue applications.

In certain medical procedures, it is desirable to have additionalmedical devices present at the anatomical location in order to provideadditional support, fixation, and/or stabilization at the locus ofrepair, replacement, and/or augmentation. Where such secondary medicaldevices are desired, the implant surface in the hydrated state can beconformable to the anatomical surface, the secondary medical devicesurface, and/or both surfaces. Examples of suitable secondary medicaldevices can include, but are not limited to, bone screws, bone plates,metallic and polymer meshes, as well as metallic and polymer plates andcaps such as those used in cranial surgeries.

The implant may have a variable range of degradation profiles that canbe manipulated to align with the clinical indication for which it isintended to be implanted. For example, when the implant is selected foruse in hernia repair, the porous body that forms the implant can have adegradation profile that substantially matches the natural tissuereplacement rate of native dura mater. In vitro degradation testing,done under conditions simulating an in vivo environment, can beperformed to evaluate an implant's degradation profile with respect to adesired clinical indication. In vitro testing can be conducted for anylength of time as is desired, for example, one day, one week, fourweeks, two months, six months, one year, or multiple years. According toone embodiment, the porous body has a one week in vitro degradationprofile (as explained in further detail below) under simulated bodyfluid (SBF) conditions in the range of about zero to about 90 percent.

According to a further embodiment, the exopolysaccharide can be ascaffold or carrier for one or more active agents. The active agent oragents can be impregnated within the porous body of exopolysaccharidethat forms the implant, coated onto a surface of the implant, and/orboth. According to one embodiment, the active agent or agents can beimpregnated within and/or coated onto the implant substantially at ornear the time of implantation (i.e., intraoperatively). In analternative embodiment, the active agent or agents can be impregnatedwithin and/or coated onto the implant prior to the time of implantation(i.e., preoperatively). In certain embodiments, more than one activeagent can be impregnated within and/or coated onto the implant, andfurther the more than one active agents can be impregnated within and/orcoated onto the implant at different time periods. For example, someactive agents can be preoperatively combined with the implant, whileother active agents can be combined intraoperatively. Active agents thatcan be utilized with the implant include any compositions suitable fortreatment at the anatomical location, such as, bone marrow, autograft,osteoinductive small molecules, osteogenic material, stem cells, bonemorphogenetic proteins, antibacterial agents, calcium phosphateceramics, and mixtures and blends thereof.

EXAMPLES

The following examples are provided to further describe some of theembodiments disclosed herein. The examples are intended to illustrate,not to limit, the disclosed embodiments.

Example 1: Microorganism and Culture Conditions

A proprietary Komagataeibacter xylinus strain from the collection ofSynthes, Inc., which has the ability to yield pellicles of above normalthicknesses for most stains of Komagataeibacter xylinus, is used in thisstudy. Other strains of K. xylinus, or other bacteria, including thoseof the genera Gluconacetobacter, Komagataeibacter, Sarcina, andAgrobacterium, can also be used. Culture media is based on varioussugars including monosaccharides (such as glucose, fructose, galactose,xylose), disaccharides (such as sucrose, maltose, lactose), sugaralcohols (such as glycerol or arabitol), or combinations thereof.Nitrogen sources in the medium are also important for pellicleuniformity and growth as well as pellicle hue. Corn steep liquor servesas the nitrogen source in the subject medium. Other sources includebacterial, vegetable, or animal peptones, waste beer yeast, cabbagejuice, and dry oil mill residues. Various salts and amino acids areadded to the medium to promote propagation and cellular metabolism. Thehomogenous medium is sterilized to mitigate contamination risk and topromote the hydrolysis of disaccharides into monosaccharides. The extentof hydrolysis is dependent on heat energy transferred during thesterilization process. Steam lethality serves as a potential measure ofheat energy. Desirable lethality ranges from 6 to 40 minutes, measureddirectly with a thermocouple, which is essential in determining accuratelethality values.

The cells for the inoculum are cultured in Erlenmeyer flasks filled withmedia for a period of 1-3 days at 30±2° C. The inoculated media withdeposited pellicle is then gently stirred and used as inoculum toconduct large-scale fermentation in tray reactors. The trays areincubated in static conditions at 30±2° C. for a period of 5-60 daysuntil a uniform pellicle with the appropriate thickness is formed on thesurface. Pellicles are harvested and purified by washing with 1-6%aqueous NaOH with 1-4 cycles for 1-5 hrs total time. Pellicles arebleached with 0.1-1% H₂O₂ for 1-2 cycles for 1-2 hrs total time followedby soaking in multiple changes of distilled water until neutral pH isreached. Finally, the pellicles are mechanically pressed to the desiredthicknesses. Pellicles are processed through either thermal modificationvia freezing and dehydration at −5° C. to −80° C. for 1-30 days orchemical treatment using, for example, acetone, methanol, ethanol, orother water miscible organic solvents. Pellicles may be γ-irradiated atthe range of 22.5-29 kGy or steam-sterilized. Additional water may beextracted by treatment with ethanol or methanol. Pellicles may beprocessed through a critical point drying system prior to proceeding tosterilization steps.

Example 2: Cloning and Expression of Escherichia Coli DerivedN-Acetylglucosamine Phosphoenolpyruvate-Dependent SugarPhosphotransferase System Permease in Komagataeibacter xylinus and theProduction of an Exopolysaccharide Containing D-Glucose andN-Acetylglucosamine

The Escherichia coli nagE gene (encoding N-acetylglucosaminephosphoenolpyruvate-dependent sugar phosphotransferase system permease,EC 2.7.1.193) is cloned into the expression vector pSEVA331 (GenBank:JX560333.1) under control of the LuxR promoter (Florea, et al 2016a). K.xylinus is transformed with the resulting plasmid as described byFlorea, et al, 2016a and 2016b. Transformed bacteria are cultured inErlenmeyer flasks filled with media for a period of 1-3 days at 30±2° C.as described in Example 1, above. The inoculated media with depositedpellicle is gently stirred and used as inoculum to conduct alarger-scale fermentation in tray reactors as described in Example 1,above, except the media in the tray reactors is further supplementedwith 0.5-2% N-acetylglucosamine and 0.01-1 μM N-acyl homoserine lactone.

Pellicles of exopolysaccharide comprising D-glucose andN-acetylglucosamine subunits are harvested and purified by washing with1-6% aqueous NaOH with 1-4 cycles for 1-5 hrs total time. Pellicles arebleached with 0.1-1% H₂O₂ for 1-2 cycles for 1-2 hrs total time followedby soaking in multiple changes of distilled water until neutral pH isreached. Finally, the pellicles are mechanically pressed to the desiredthicknesses.

The relative amounts of D-glucose and N-acetylglucosamine in theresulting exopolysaccharide is determined by LC-MS/MS after acidhydrolysis as described by Yadav, et al, 2010.

Example 3: Cloning and Expression of Escherichia coli DerivedN-Acetylglucosamine Phosphoenolpyruvate-Dependent SugarPhosphotransferase System Permease and Candida albican DerivedN-Acetylglucosamine Metabolism Genes in Komagataeibacter xylinus and theProduction of an Exopolysaccharide Containing D-Glucose andN-Acetylglucosamine

The Escherichia coli nagE gene, Candida albicans nag5 gene (encodingN-acetylglucosamine kinase, EC:2.7.1.59), Candida albicans agm1 gene(encoding phosphoacetylglucosamine mutase, EC:5.4.2.3), and Candidaalbicans uap1 gene (encoding UDP-N-acetylglucosamine pyrophosphorylase,EC 2.7.7.23) are cloned into the expression vector pSEVA331 (GenBank:JX560333.1) under control of the LuxR promoter (Florea, et al 2016a). K.xylinus is transformed with the resulting plasmid as described byFlorea, et al, 2016a and 2016b. Transformed bacteria are cultured inErlenmeyer flasks filled with media for a period of 1-3 days at 30±2° C.as described in Example 1, above. The inoculated media with depositedpellicle is gently stirred and used as inoculum to conduct alarger-scale fermentation in tray reactors as described in Example 1,above, except the media in the tray reactors is further supplementedwith 0.5-2% N-acetylglucosamine and 0.01-1 μM N-acyl homoserine lactone.

Pellicles of exopolysaccharide comprising D-glucose andN-acetylglucosamine subunits are harvested and purified by washing with1-6% aqueous NaOH with 1-4 cycles for 1-5 hrs total time. Pellicles arebleached with 0.1-1% H₂O₂ for 1-2 cycles for 1-2 hrs total time followedby soaking in multiple changes of distilled water until neutral pH isreached. Finally, the pellicles are mechanically pressed to the desiredthicknesses.

The relative amounts of D-glucose and N-acetylglucosamine in theresulting exopolysaccharide is determined by LC-MS/MS after acidhydrolysis as described by Yadav, et al, 2010.

Example 4: Cloning and Expression of Escherichia coli DerivedN-Acetylglucosamine Phosphoenolpyruvate-Dependent SugarPhosphotransferase System Permease and Escherichia coli and Mus musculusDerived N-Acetylglucosamine Metabolism Genes in Komagataeibacter xylinusand the Production of an Exopolysaccharide Containing D-Glucose andN-Acetylglucosamine

The Escherichia coli nagE gene, Escherichia coli nagK gene (encodingGlcNac kinase), Mus musculus pgm3 gene (encodingphosphoacetyl-glucosamine mutase, EC:5.4.2.3), and Escherichia coli glmUgene (encoding bifunctional UDP-N-acetylglucosaminepyrophosphorylase/Glucosamine-1-phosphate N-acetyltransferase,EC:2.7.7.23 and 2.3.1.157) are cloned into the expression vectorpSEVA331 (GenBank: JX560333.1) under control of the LuxR promoter(Florea, et al 2016a). K. xylinus is transformed with the resultingplasmid as described by Florea, et al, 2016a and 2016b. Transformedbacteria are cultured in Erlenmeyer flasks filled with media for aperiod of 1-3 days at 30±2° C. as described in Example 1, above. Theinoculated media with deposited pellicle is gently stirred and used asinoculum to conduct a larger-scale fermentation in tray reactors asdescribed in Example 1, above, except the media in the tray reactors isfurther supplemented with 0.5-2% N-acetylglucosamine and 0.01-1 μMN-acyl homoserine lactone.

Pellicles of exopolysaccharide comprising D-glucose andN-acetylglucosamine subunits are harvested and purified by washing with1-6% aqueous NaOH with 1-4 cycles for 1-5 hrs total time. Pellicles arebleached with 0.1-1% H₂O₂ for 1-2 cycles for 1-2 hrs total time followedby soaking in multiple changes of distilled water until neutral pH isreached. Finally, the pellicles are mechanically pressed to the desiredthicknesses.

The relative amounts of D-glucose and N-acetylglucosamine in theresulting exopolysaccharide is determine by LC-MS/MS after acidhydrolysis as described by Yadav, et al, 2010.

Example 5: Supercritical CO₂ (sCO₂) Drying

Cellulose sheets are extracted with ethanol or methanol by conducting astepwise extraction: 30%, 50%, 90% (v/v) and absolute ethanol for 3hours at each percentage. Samples are then dried using a Speed SFEsupercritical CO₂ extraction system (Applied Separations, Inc.,Allentown, Pa.) to maintain its open three-dimensional structure andnano-porosity.

Example 6: Manufacture of Resorbable Microbial-Derived Exopolysaccharide

This example is directed to a preparation of modified microbial-derivedexopolysaccharide films produced by K. xylinus within a controlledenvironment to minimize bioburden (microorganism contamination). From apropagation vessel, sterilized media may be inoculated with K. xylinusexpressing one or more of the exogenous enzymes described herein, filledinto bioreactor trays and incubated until optimal growth of the pellicleis observed. The pellicles may be removed from the trays and then mayundergo caustic chemical processing (depyrogenation) in a tank for aboutone hour. The pellicles then undergo a continuous rinse with filteredwater. The films are compressed within a pneumatic press to yield apellicle having a weight of approximately 4 to 10 g. Each unit is placedin a pouch, sealed, sterilized, and used for various tests, inclusive ofexopolysaccharide content, endotoxin (LAL), and mechanical strength.

Example 7: Manufacture of Implantable Resorbable Microbial-DerivedExopolysaccharide for Hernia Mesh

This example is directed to a preparation of modified microbial-derivedexopolysaccharide films per the initial steps of Examples 1 and 2.Following chemical processing, the films are compressed within apneumatic press to yield a pellicle having a weight of approximately 9to 13 g. Each unit is placed in a closed container and decreased intemperature to below 0° C. After at least 24 hours, the temperature isincreased to above freezing and excess moisture that is released isdecanted to partially dehydrate the exopolysaccharide. The partiallydehydrated material is placed in a pouch, sealed, sterilized, and usedfor various tests, inclusive of exopolysaccharide content, endotoxin(LAL), and mechanical strength.

It will be appreciated by those skilled in the art that variousmodifications and alterations of the invention can be made withoutdeparting from the broad scope of the appended claims. Some of thesehave been discussed above and others will be apparent to those skilledin the art.

The disclosures of each patent, patent application, and publicationcited or described in this document are hereby incorporated herein byreference, in its entirety.

EMBODIMENTS

The following list of embodiments is intended to complement, rather thandisplace or supersede, the previous descriptions.

Embodiment 1

A microorganism for the production of an exopolysaccharide, wherein:

-   -   a. the microorganism expresses an exogenous enzyme, wherein the        exogenous enzyme is N-acetylglucosamine        phosphoenolpyruvate-dependent sugar phosphotransferase system        permease or a biologically active homologue thereof; and    -   b. the microorganism produces an exopolysaccharide that        comprises N-acetylglucosamine and D-glucose.

Embodiment 2

The microorganism of embodiment 1, wherein the microorganism furtherexpresses at least one exogenous enzyme involved in N-acetylglucosamineuptake or metabolism.

Embodiment 3

The microorganism of embodiment 2, wherein the at least one exogenousenzyme involved in N-acetylglucosamine uptake or metabolism comprisesone or more of N-acetylglucosamine kinase, phosphoacetyl-glucosaminemutase, or UDP-N-acetylglucosamine pyrophosphorylase.

Embodiment 4

The microorganism of embodiment 3, wherein the UDP-N-acetylglucosaminepyrophosphorylase also has glucosamine-1-phosphate N-acetyltransferaseactivity.

Embodiment 5

The microorganism of embodiment 3 or 4, wherein the N-acetylglucosaminekinase and/or the UDP-N-acetylglucosamine pyrophosphorylase are derivedfrom a source other than yeast.

Embodiment 6

The microorganism of any one of the preceding embodiments, wherein theexogenous enzyme is encoded by a nucleic acid.

Embodiment 7

The microorganism of embodiment 6, wherein the nucleic acid is a cDNAmolecule.

Embodiment 8

The microorganism of embodiment 6 or 7, wherein the nucleic acid iscontained in a recombinant vector.

Embodiment 9

The microorganism of embodiment 8, wherein the recombinant vector is aviral or a plasmid vector.

Embodiment 10

The microorganism of any one of embodiments 6-9, wherein the nucleicacid is operably linked to a promoter.

Embodiment 11

The microorganism of embodiment 10, wherein the promoter is aconstitutive or inducible promoter.

Embodiment 12

The microorganism of any one of the preceding embodiments, wherein themicroorganism is a bacterium.

Embodiment 13

The microorganism of embodiment 12, wherein the bacterium belongs to thefamily Acetobacteraceae or a genus comprising Agrobacterium, Aerobacter,Achromobacter, Azotobacter, Rhizobium, Sarcina, or Salmonella.

Embodiment 14

The microorganism of embodiment 13, wherein the bacterium isKomagataeibacter xylinus.

Embodiment 15

A bacterium for the production of an exopolysaccharide, wherein:

-   -   a. the bacterium expresses at least one exogenous enzyme,        wherein the at least one exogenous enzyme is N-acetylglucosamine        kinase, yeast-derived phosphoacetyl-glucosamine mutase, or        UDP-N-acetylglucosamine pyrophosphorylase; and    -   b. the bacterium produces an exopolysaccharide that comprises        N-acetylglucosamine and D-glucose; and    -   wherein        -   the N-acetylglucosamine kinase and the            UDP-N-acetylglucosamine pyrophosphorylase are derived from a            source other than yeast, or        -   the N-acetylglucosamine kinase is derived from a source            other than yeast and the UDP-N-acetylglucosamine            pyrophosphorylase is derived from yeast, or        -   the N-acetylglucosamine kinase is derived from yeast and the            UDP-N-acetylglucosamine pyrophosphorylase is derived from a            source other than yeast.

Embodiment 16

The bacterium of embodiment 15, wherein the at least one exogenousenzyme is encoded by a nucleic acid.

Embodiment 17

The bacterium of embodiment 16, wherein the nucleic acid is a cDNAmolecule.

Embodiment 18

The bacterium of embodiment 16 or 17, wherein the nucleic acid iscontained in a recombinant vector.

Embodiment 19

The bacterium of embodiment 18, wherein the recombinant vector is aviral or a plasmid vector.

Embodiment 20

The bacterium of any one of embodiments 16-19, wherein the nucleic acidis operably linked to a promoter.

Embodiment 21

The bacterium of embodiment 20, wherein the promoter is a constitutiveor inducible promoter.

Embodiment 22

The bacterium of any one of embodiments 15-21, wherein the bacteriumbelongs to the family Acetobacteraceae or a genus comprisingAgrobacterium, Aerobacter, Achromobacter, Azotobacter, Rhizobium,Sarcina, or Salmonella.

Embodiment 23

The bacterium of embodiment 22, wherein the bacterium isKomagataeibacter xylinus.

Embodiment 24

A method for producing an exopolysaccharide comprising:

-   -   a. culturing a microorganism under conditions effective to        produce the exopolysaccharide, and    -   b. recovering the exopolysaccharide,    -   wherein said microorganism expresses an exogenous enzyme,        wherein the exogenous enzyme is N-acetylglucosamine        phosphoenolpyruvate-dependent sugar phosphotransferase system        permease or a biologically active homologue thereof, and wherein        the exopolysaccharide comprises N-acetylglucosamine and        D-glucose.

Embodiment 25

The method of embodiment 24, wherein the microorganism further expressesat least one exogenous enzyme involved in N-acetylglucosamine uptake andmetabolism.

Embodiment 26

The method of embodiment 25, wherein the at least one exogenous enzymeinvolved in N-acetylglucosamine uptake and metabolism comprises one ormore of N-acetylglucosamine kinase, phosphoacetyl-glucosamine mutase, orUDP-N-acetylglucosamine pyrophosphorylase.

Embodiment 27

The method of embodiment 26, wherein the UDP-N-acetylglucosaminepyrophosphorylase also has glucosamine-1-phosphate N-acetyltransferaseactivity.

Embodiment 28

The method of embodiment 26 or 27, wherein the N-acetylglucosaminekinase and/or the UDP-N-acetylglucosamine pyrophosphorylase are derivedfrom a source other than yeast.

Embodiment 29

The method of any one of embodiments 24 to 28, wherein the microorganismis a bacterium.

Embodiment 30

The method of embodiment 29, wherein the bacterium belongs to the familyAcetobacteraceae or a genus comprising Agrobacterium, Aerobacter,Achromobacter, Azotobacter, Rhizobium, Sarcina, or Salmonella.

Embodiment 31

The method of embodiment 30, wherein the bacterium is Komagataeibacterxylinus.

Embodiment 32

A method for producing an exopolysaccharide comprising:

-   -   a. culturing a bacterium under conditions effective to produce        the exopolysaccharide, and    -   b. recovering the exopolysaccharide,    -   wherein said bacterium expresses at least one exogenous enzyme,        wherein the at least one exogenous enzyme is N-acetylglucosamine        kinase, yeast-derived phosphoacetyl-glucosamine mutase, or        UDP-N-acetylglucosamine pyrophosphorylase, wherein the        exopolysaccharide comprises N-acetylglucosamine and D-glucose,        and wherein    -   the N-acetylglucosamine kinase and the UDP-N-acetylglucosamine        pyrophosphorylase are derived from a source other than yeast, or    -   the N-acetylglucosamine kinase is derived from a source other        than yeast and the UDP-N-acetylglucosamine pyrophosphorylase is        derived from yeast, or    -   the N-acetylglucosamine kinase is derived from yeast and the        UDP-N-acetylglucosamine pyrophosphorylase is derived from a        source other than yeast.

Embodiment 33

The method of embodiment 32, wherein the bacterium belongs to the familyAcetobacteraceae or a genus comprising Agrobacterium, Aerobacter,Achromobacter, Azotobacter, Rhizobium, Sarcina, or Salmonella.

Embodiment 34

The method of embodiment 33, wherein the bacterium is Komagataeibacterxylinus.

Embodiment 35

The method of any one of embodiments 24-34, wherein the microorganism orbacterium is cultured in a culture medium containing D-glucose,N-acetylglucosamine, glucosamine, 3-O-methylglucose, 2-deoxyglucose, ora combination thereof.

Embodiment 36

The method of any one of embodiments 24-35, further comprising the stepof at least partially dehydrating the body of the exopolysaccharide.

Embodiment 37

The method of embodiment 36, wherein the step of at least partiallydehydrating the body of the exopolysaccharide is performed by mechanicalpressing.

Embodiment 38

The method of embodiment 36, wherein the step of at least partiallydehydrating the body of the exopolysaccharide is performed by criticalpoint drying using supercritical carbon dioxide.

Embodiment 39

The method of any one of embodiments 24-38, further comprising the stepof contacting the exopolysaccharide with one or more active agents.

Embodiment 40

The method of any one of embodiments 36-39, further comprising the stepof reacting the exopolysaccharide with an oxidizing agent.

Embodiment 41

The method of embodiment 40, wherein the oxidizing agent comprisesmetaperiodate, hypochlorite, dichromate, peroxide, permanganate,nitrogen dioxide, or a combination thereof.

Embodiment 42

The method of embodiment 41, wherein the oxidizing agent is sodiummetaperiodate.

Embodiment 43

An implant for soft tissue repair, replacement, or augmentationcomprising the exopolysaccharide produced by the method of any one ofembodiments 24-42.

Embodiment 44

The implant of embodiment 43 for use in hernia repair, repair of duramater, replacement of dura mater, or any combination thereof.

What is claimed:
 1. A bacterium for the production of anexopolysaccharide comprising N-acetylglucosamine and D-glucose, wherein:the bacterium belongs to a genus comprising Komagataeibacter,Agrobacterium, Aerobacter, Achromobacter, Azotobacter, Rhizobium,Sarcina, or Salmonella and the bacterium expresses a. an Escherichiacoli N-acetylglucosamine phosphoenolpyruvate-dependent sugarphosphotransferase system permease; and b. an Escherichia coliN-acetylglucosamine kinase, a Mus musculus phosphoacetyl-glucosaminemutase, and an Escherichia coli bifunctional UDP-N-acetylglucosaminepyrophosphorylase/glucosamine-1-phosphate N-acetyltransferase, and thebacterium produces an exopolysaccharide that comprisesN-acetylglucosamine and D-glucose.
 2. The bacterium of claim 1, whereinthe bacterium is Komagataeibacter xylinus.